Bottom Line:
The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases.The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51.Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

ABSTRACTThe FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

pone-0057508-g006: Influence of FKBP inhibitors.A Purified proteins were mixed and treated with DMSO or rapamycin (1 µM). After 3 h a GST-pull-down was performed followed by Western blotting. B HEK293T cells were transfected with the indicated constructs. After 48 h 1 µM FK1706 was added for 1 h. The lysates were immunoprecipitated and a Western blot was performed. C and D HEK293T cells were transfected with FLAG-FKBP51 and HA-tagged PHLPP1 or HA-tagged PHLPP2. After 48 hFKBP inhibitors (1 µM) or DMSO were added for 1h. Lysates were immunoprecipitated and analyzed by Western blotting. E HEK293T cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt phosphorylation was determined using a homogeneous time- resolved FRET assay. F SHSY-5Y cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt and mTOR phosphorylation was determined using a homogeneous time resolved FRET assay. G SU.86.86 cells were plated, treated with Gemcitabine in the absence or presence of FK1706. Cell survival relative to DMSO treated controls was determined.

Mentions:
The ability of several FKBP members to bind to Akt (Figure 1) suggested the FK506-binding pocket common to all these proteins as an interaction site. We therefore tested if FKBP ligands blocking the PPIase domain can reduce binding of Akt to FKBP51. We first performed a pull-down experiment using purified FKBP51 and purified AktS473D as bait in the absence and presence of the high-affinity ligand rapamycin. The amount of FKBP51 that was specifically retained by Akt was not affected by an excess of rapamycin (Figure 6A). We next co-immunoprecipitated Akt with FKBP51 or its TPR-mutant in the presence or absence of the non-immunosuppressive FK506 analog FK1706 [34]. Binding of Akt was slightly reduced for the TPR-mutant but it was still significantly retained compared to background (Figure 6B). The interaction with neither FKBP51 construct was affected by the treatment with FK1706. Similar results were obtained in cells treated with FK506 or rapamycin (Figure S2). Since PHLPP is regulating Akt phosphorylation and is proposed to be part of the Akt-FKBP51-PHLPP complex [23] we explored whether FKBP inhibitors affected the FKBP51-PHLPP complex. FKBP inhibitors had no effect on the integrity of the complex of FKBP51 with PHLPP1 or PHLPP2 (Figure 6C, Figure 6D). Finally, we tested whether cellular Akt or mTOR phosphorylation would be affected by FKBP inhibitors. Neither the phosphorylation of Akt at T308 nor S473 was affected in HEK293T cells treated with high concentrations of FK1706. Under the same conditions the mTOR inhibitor Torin-1 reduced Akt phosphorylation at both sites [35], while the ATP-competitive inhibitor AT7867 enhanced it demonstrating that the assay was able to detect the dynamic regulation of Akt in these cells (Figure 6E). Similar results were obtained for Akt S473 and mTOR S2448 phosphorylation in FK1706 or FK506-treated SHSY-5Y (Figure 6F) and HeLa cells (Figure S3). Rapamycin which served as control stimulated and inhibited both phosphorylations in the expected way. Since FKBP51 was shown to regulate the sensitivity of pancreatic cancer cells to chemotherapeutics [23], [24] we tested the effect of FKBP inhibitors in these cells. In a cell viability assay we observed that FK1706 (10 µM) did not enhance the cytotoxic effect of Gemcitabine in SU.86.86 cells (Figure 6G).

pone-0057508-g006: Influence of FKBP inhibitors.A Purified proteins were mixed and treated with DMSO or rapamycin (1 µM). After 3 h a GST-pull-down was performed followed by Western blotting. B HEK293T cells were transfected with the indicated constructs. After 48 h 1 µM FK1706 was added for 1 h. The lysates were immunoprecipitated and a Western blot was performed. C and D HEK293T cells were transfected with FLAG-FKBP51 and HA-tagged PHLPP1 or HA-tagged PHLPP2. After 48 hFKBP inhibitors (1 µM) or DMSO were added for 1h. Lysates were immunoprecipitated and analyzed by Western blotting. E HEK293T cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt phosphorylation was determined using a homogeneous time- resolved FRET assay. F SHSY-5Y cells were stimulated with FCS for 1 h in the presence of the indicated compounds. After cell lysis, cellular Akt and mTOR phosphorylation was determined using a homogeneous time resolved FRET assay. G SU.86.86 cells were plated, treated with Gemcitabine in the absence or presence of FK1706. Cell survival relative to DMSO treated controls was determined.

Mentions:
The ability of several FKBP members to bind to Akt (Figure 1) suggested the FK506-binding pocket common to all these proteins as an interaction site. We therefore tested if FKBP ligands blocking the PPIase domain can reduce binding of Akt to FKBP51. We first performed a pull-down experiment using purified FKBP51 and purified AktS473D as bait in the absence and presence of the high-affinity ligand rapamycin. The amount of FKBP51 that was specifically retained by Akt was not affected by an excess of rapamycin (Figure 6A). We next co-immunoprecipitated Akt with FKBP51 or its TPR-mutant in the presence or absence of the non-immunosuppressive FK506 analog FK1706 [34]. Binding of Akt was slightly reduced for the TPR-mutant but it was still significantly retained compared to background (Figure 6B). The interaction with neither FKBP51 construct was affected by the treatment with FK1706. Similar results were obtained in cells treated with FK506 or rapamycin (Figure S2). Since PHLPP is regulating Akt phosphorylation and is proposed to be part of the Akt-FKBP51-PHLPP complex [23] we explored whether FKBP inhibitors affected the FKBP51-PHLPP complex. FKBP inhibitors had no effect on the integrity of the complex of FKBP51 with PHLPP1 or PHLPP2 (Figure 6C, Figure 6D). Finally, we tested whether cellular Akt or mTOR phosphorylation would be affected by FKBP inhibitors. Neither the phosphorylation of Akt at T308 nor S473 was affected in HEK293T cells treated with high concentrations of FK1706. Under the same conditions the mTOR inhibitor Torin-1 reduced Akt phosphorylation at both sites [35], while the ATP-competitive inhibitor AT7867 enhanced it demonstrating that the assay was able to detect the dynamic regulation of Akt in these cells (Figure 6E). Similar results were obtained for Akt S473 and mTOR S2448 phosphorylation in FK1706 or FK506-treated SHSY-5Y (Figure 6F) and HeLa cells (Figure S3). Rapamycin which served as control stimulated and inhibited both phosphorylations in the expected way. Since FKBP51 was shown to regulate the sensitivity of pancreatic cancer cells to chemotherapeutics [23], [24] we tested the effect of FKBP inhibitors in these cells. In a cell viability assay we observed that FK1706 (10 µM) did not enhance the cytotoxic effect of Gemcitabine in SU.86.86 cells (Figure 6G).

Bottom Line:
The FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases.The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51.Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.

ABSTRACTThe FK506-binding protein 51 (FKBP51) is an Hsp90-associated co-chaperone which regulates steroid receptors and kinases. In pancreatic cancer cell lines, FKBP51 was shown to recruit the phosphatase PHLPP to facilitate dephosphorylation of the kinase Akt, which was associated with reduced chemoresistance. Here we show that in addition to FKBP51 several other members of the FKBP family bind directly to Akt. FKBP51 can also form complexes with other AGC kinases and mapping studies revealed that FKBP51 interacts with Akt via multiple domains independent of their activation or phosphorylation status. The FKBP51-Akt1 interaction was not affected by FK506 analogs or Akt active site inhibitors, but was abolished by the allosteric Akt inhibitor VIII. None of the FKBP51 inhibitors affected AktS473 phosphorylation or downstream targets of Akt. In summary, we show that FKBP51 binds to Akt directly as well as via Hsp90. The FKBP51-Akt interaction is sensitive to the conformation of Akt1, but does not depend on the FK506-binding pocket of FKBP51. Therefore, FKBP inhibitors are unlikely to inhibit the Akt-FKBP-PHLPP network.